Transgenic microalgae and use thereof for oral delivery of proteins

ABSTRACT

Transgenic microalgae expressing at least one exogenous biologically active protein. The protein-expressing microalgae are used for the oral delivery of the biologically active protein to the target organism in its intact and functional form. The exogenous protein, expressed in algae, is characterized by being biologically active, exerting at least one specific activity having a beneficial effect on the subject consuming the algae. The transgenic microalgae are used as animal food for aquatic or land animals welfare or as food supplement for human healthcare.

FIELD OF THE INVENTION

The present invention relates to transgenic microalgae expressingexogenous biologically active proteins and use thereof for oral deliveryof the biologically active proteins to animals and humans.

BACKGROUND OF THE INVENTION

Bioactive proteins are essential for the function of cells of livingorganisms, and are responsible for most of the activities of the cellincluding catalysis of metabolic processes, communication, defense,movement, and transport. Oral delivery of bioactive proteins istypically required for two principal purposes: delivery of therapeuticproteins and delivery of proteins that have nutritional or anotherbeneficial effect on animals, including humans.

The growing demand for food over the world, particularly for animalprotein, together with the awareness towards environmental impacts ofanimal growth, requires the development of sophisticated agriculturalmanagement tools that would improve the productivity and weight gain ofterrestrial as well as aquatic farm animals. Development of aquaculturesfor the growth of marine animals including fish, crustaceans andmollusks is of particular importance, as marine animal are considered asan healthier source for proteins.

However, aquaculture is still not a completely efficient system forgrowing marine animals and fish in particular. Typically, fish require arelatively long period of time to reach an acceptable size and weight.Furthermore, many breeds of fish mature in an inefficient manner, suchthat problems may occur in the fish population, resulting in loss of aportion of the population due to excessively slow growth, poormorphology (for ornamental fish) and so forth. Furthermore, in theartificial aquaculture environment, the aquatic animals are moresusceptible to infectious diseases and, when disease does occur, it canspread rapidly through entire populations with high mortality.

There is thus a need for routes to administer therapeutic or nutritionalproteins to terrestrial as well as aquatic farm animals that are notcostly and do not require laborious efforts. Oral delivery oftherapeutic or nutritional proteins to humans is also highly desirable,as this mode of administration does not require professional manpowerand significantly increases the patient compliance to the prescribeddose. The biological activity of a protein depends on its sequenceand/or conformation, which must be preserved until the protein reachesits target of activity. For successful oral delivery, proteins should beprotected from chemical and enzymatic degradation that may occur duringprocessing the proteins into food or a feed composition and through thedelivery via the animal's gastrointestinal tract. In addition, theprotein should overcome structural barriers that preclude entry into theanimal or access to the target destination.

Microalgae (single cell alga or phytoplankton) represent the largest,but most poorly understood, kingdom of microorganisms on the earth. Likeplants are to terrestrial animals, the microalgae represent the naturalnutritional base and primary source of all the phytonutrients in theaquatic food chain. Expression of recombinant proteins in algae has beenreported, and various methods are available for production of exogenousproteins within the algae cells, particularly within the cell plastid.International (PCT) Application Publication No. WO 2011/063284 disclosesmethods of expressing therapeutic proteins in photosynthetic organisms,including prokaryotes such as cyanobacteria and eukaryotes such as algaand plants. Transformation of eukaryotes is preferably into the plastidgenome, typically into the chloroplast genome. The Application disclosesexpression of particular therapeutic proteins within algae cells.

Various attempts have been made to use microalgae as delivery means forproteins. For example, International (PCT) Application Publication No.WO 01/98335 discloses delivery systems and methods for delivering abiologically active protein to a host animal. The systems and methodsprovided include obtaining an algal cell transformed by an expressionvector, the expression vector comprising a nucleotide sequence codingfor the biologically active protein, operably linked to a promoter. Inone illustrated embodiment, the biologically active protein is anantigenic epitope and upon administration to the animal the algal cellinduces an immune response in the host animal.

International (PCT) Application Publication No. WO 2002/076391 disclosesthe use of microbial cells which are used as feed components inaquaculture or agriculture, and which also contain exogenous peptides,proteins, and/or antibodies, which will convey resistance or immunity toviral or bacterial pathogens or otherwise improve the health andperformance of the species consuming said microbial cells. The microbialcells can be yeast, fungi, bacteria, or algae. The proteins and/orantibodies may be expressed inside the microbial cells by direct geneticmodification of the microbe itself, or by the infection of the microbewith a virus that has been altered to express the protein of interest.

International (PCT) Application Publication No. WO 2008/027235 disclosesmethods for prevention, amelioration or treatment of a disease ordisorder in an aquatic animal, by feeding the aquatic animal directly orindirectly with genetically modified micro algae that expresses arecombinant molecule that specifically targets one or more key epitopesof a pathogen that infects the aquatic animal.

U.S. Patent Application Publication No. 2011/0014708 discloses method ofproducing a foreign desired gene product in algae that comprisesweakening or removing the algae cell wall by a protein enzyme solutionto facilitate the gene transfer and a feed composition comprising thetransgenic algae or its offspring. The invention also provides amodified nucleic acid for expressing bovine lactoferricin (LFB) inalgae.

However, there is still an unmeet need for and it would be highlyadvantageous to have an oral delivery system that is easy for productionand use, maintains the biological activity of the protein and facilitateabsorption of the biologically active protein systemically.

SUMMARY OF THE INVENTION

The present invention provides an algal based platform for oral deliveryof biologically active proteins to an organism, providing for thesystemic absorption of the biologically active protein in its activeform. Particularly, the present invention provides transgenic microalgaeexpressing at least one exogenous protein within a predeterminedsubcellular compartment. The protein-expressing microalgae are used asanimal food or food additive applicable for feeding aquatic animals andland animals as well as for food supplement for humans. The exogenousprotein is characterized by being biologically active, exerting at leastone specific activity having a beneficial effect on the target organism.

The present invention is based in part on the unexpected discovery thatthe expressed protein remains active and exerts its specific activityhaving a beneficial effect when the algae are orally consumed by aquaticanimals (including fish and crustaceans) as well as by land animals(including mice and poultry). Typically, the protein remains in itsintact form. Without wishing to be bound by any particular theory ormechanism of action, the preserved protein activity may be attributed toits localization within the intact microalga, such that the microalgalcell serves as a natural encapsulation material protecting the proteinfrom being degraded in the animal's gastrointestinal tract and/or acidicstomach. According to certain typical embodiments of the presentinvention, the protein is expressed in a microalgal subcellularcompartment, particularly in the vacuole.

Thus, according to one aspect, the present invention provides atransgenic eukaryotic microalga comprising an expression cassettecomprising at least one transcribable polynucleotide encoding abiologically active exogenous protein, wherein the biologically activeexogenous protein is expressed within a subcellular compartment of themicroalga cell.

The subcellular compartment in which the protein is expressed depends onthe microalga species, the type of the protein expressed and the animalspecies to be fed. According to certain embodiments, the subcellularcompartment is selected from the group consisting of vacuole,endoplasmic reticulum, Golgi system, lysosome and peroxisome. Eachpossibility represents a separate embodiment of the present invention.

According to certain exemplary embodiments, the exogenous protein isexpressed within the microalga cell vacuole. According to theseembodiments, the expression cassette further comprises a polynucleotideencoding a vacuole targeting peptide. According to some embodiments, thepeptide targeting the exogenous protein into the vacuole is a shortvacuole leader sequence having the amino acid sequence set forth in SEQID NO:4. According to other embodiments, the polynucleotide encoding theshort vacuole leader peptide comprises the nucleic acid sequence setforth in SEQ ID NO:18.

According to certain other exemplary embodiments, the exogenous proteinis expressed within the microalga cell endoplasmic reticulum (ER).According to these embodiments, the expression cassette furthercomprises a polynucleotide encoding an ER targeting peptide. Accordingto some embodiments, the peptide targeting the exogenous protein intothe ER is a Phaeodactylum tricornutum endoplasmic reticulum (Bip) leadersequence having the amino acid sequence set forth in SEQ ID NO:2.According to other embodiments, the polynucleotide encoding the ERleader peptide comprises the nucleic acid sequence set forth in SEQ IDNO:16.

Various microalgae species can be used according to the teachings of thepresent invention. According to certain embodiments, the microalga usedaccording to the teachings of the present invention is a marinemicroalga. According to certain embodiments, the microalga is selectedfrom the group consisting of, but not restricted to, Phaeodactylumtricornutum; Dunaliella spp.; Nannochloropsis spp. includingNannochloropsis oculata, Nannochloropsis salina, Nannochloropsisgaditana; Nannochioris spp., Tetraselmis spp. Including Tetraselmissuecica, Tetraselmis chuii; lsochtysis galbana; Pavlova spp.; Amphiprorahyaline; Chaetoceros muelleri; and Neochloris oleoabundans. Eachpossibility represents a separate embodiment of the present invention.

According to certain specific embodiments, the microalga is selectedfrom the group consisting of Phaeodactylum tricornutum, Nannochlorisspp., Nannochloropsis spp. and Dunaliella spp.

According to other specific embodiments, the microalga is Phaeodactylumtricornutum.

The transgenic microalgae of the present invention can be transformed toexpress any protein having an effect on the target animal consumingsame.

According to certain embodiments, the molecular weight of the expressedprotein is up to 150 kDa. According to other embodiments, the molecularweight of the expressed protein is up to 140 kDa, 130 kDa, 120 kDa or110 kDa. According to other embodiments the molecular weight of theexpressed protein is in the range of 1-100 kDa. According to certainexemplary embodiments the molecular weight if the expressed protein isin the range of 1-50 kDa.

According to certain embodiments, the protein has a beneficial effect onat least one of the growth, development and survival of the consuminganimal. Each possibility represents a separate embodiment of the presentinvention. According to other embodiments, the protein has a therapeuticeffect on the animal consuming the transgenic microalgae.

According to yet additional embodiments, the animal is an aquatic animaland the expressed protein affects the aquatic animal morphology.According to some embodiments, the protein has an effect of the aquaticbody deformation. Body deformation includes but is not limited to anymorphological irregularity, any type of body asymmetry, irregular bodyshape, irregular fin shape, irregular tail shape; irregular body/finarea, length or width ratios; irregular body/tail area, length or widthratios; irregular tail/fin area, length or width ratios; orirregularities in any body part. According to certain currently specificembodiments, the expressed protein reduces or eliminates bodydeformation of the aquatic animal.

According to further certain embodiments, the transgenic microalgaexpresses a hormone. According to some embodiments, the animal is anaquatic animal and the hormone is selected from the group consisting ofa growth hormone, appetite inducing hormone and spawning hormone. Eachpossibility represents a separate embodiment of the present invention.

According to certain currently specific embodiments, the expressedprotein is fish growth hormone. According to some embodiments, the fishgrowth hormone is Salmon growth hormone having the amino acid sequenceset forth in SEQ ID NO:12. According to certain specific embodiments,the fish growth hormone is encoded by the polynucleotide having thenucleic acid sequence set forth in SEQ ID NO:1.

According to additional embodiments, the expressed protein is spawninghormone having the amino acid sequence set forth in SEQ ID NO:24(Accession No.: P68072). According to certain specific embodiments, thespawning hormone is encoded by the polynucleotide having the nucleicacid sequence set forth in SEQ ID NO:25.

According to yet further embodiments, the expressed protein is appetiteinducing hormone having the amino acid sequence set forth in SEQ IDNO:20. According to certain specific embodiments, the appetite hormoneis encoded by the polynucleotide having the nucleic acid sequence setforth in SEQ ID NO:21.

According to additional aspect, the present invention provides an ediblecomposition comprising transgenic microalgae of the present invention.According to some embodiments, the edible composition is an animal foodcomposition. According to exemplary embodiments, the animal foodcomposition is for feeding aquatic animals. According to yet otherembodiments, the edible composition is for human consumption. Asdescribed hereinabove, the edible composition is used for oral deliveryof biologically active proteins. According to some embodiments,particularly when the edible composition is for animal consumption, theedible composition consists essentially of the transgenic algae.According to further embodiments, the edible composition consists of thetransgenic algae. The edible composition may be used per se or as anadditive to animal or human food.

According to an additional aspect the present invention provides amethod of delivering a biologically active protein to an animal, themethod comprising orally administering to an animal subject transgeniceukaryotic microalgae comprising an expression cassette comprising atleast one transcribable polynucleotide encoding the biologically activeprotein, wherein said biologically active protein is expressed within asubcellular compartment of the microalga cell.

According to certain embodiments, the subcellular compartment isselected from the group consisting of vacuole, endoplasmic reticulum,Golgi system, lysosome and peroxisome. Each possibility represents aseparate embodiment of the present invention.

According to certain specific embodiments, the exogenous protein isexpressed within the microalga cell vacuole. According to other specificembodiments the exogenous protein is expressed within the microalga cellendoplasmic reticulum.

According to some embodiments, the transgenic eukaryotic microalgae areadministered within an animal or human food composition.

According to a further aspect the present invention provides a methodfor improving at least one of the animal growth rate, growth pattern,reproductive health status, survival or any combination thereof,comprising administering to the animal an effective amount of thetransgenic microalgae of the present invention or a compositioncomprising same, thereby improving the growth rate and/or the growthpattern and/or the survival and/or the reproductive health status ofsaid animal.

According to certain embodiments the animal subject is a land animal oran aquatic animal. The land animal may optionally be any animal grownfor food or for a non-food purpose (the latter including but not limitedto work animals, pets and the like), including but not limited to cows,pigs, horses, dogs, cats, mice, rats, rabbits, guinea pigs, poultry andthe like. The aquatic animal may optionally be any animal grown for foodor for a non-food purpose (the latter including but not limited toornamental, and the like), including but not limited to fish,crustaceans, and corals. According to additional embodiments the animalsubject is a human.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a scheme of the complete pPhaT1 expression vector.

FIG. 2 shows Western blot of proteins extracted from transgenic algae ofthe species Phaeodactylum tricornutum expressing Salmon growth hormonetargeted to different sub-cellular organelles.

FIG. 3 shows the total growth performance of Angel fish fed with fishcommercial regular food comprising transgenic algae overexpressing fishgrowth hormone (fGH) targeted to the ER (356) or to the vacuole (398),compared to fish fed with the regular fish food (control). Fish growthis presented as percentage of weight addition relative to the initialfish weight.

FIG. 4 shows that feeding fish with regular food containing vacuoletargeted-fGH expressing algae (398) resulted in an increase in thenumber of fish reaching over 2 gr compared to fish fed with regular fishfood.

FIG. 5 shows the effect of transgenic algae overexpressing fGHadministered as food supplement on the total weight of Angel fish.398—Regular fish feed comprising 4% of vacuole targeted-fGH expressingalgae. Control—regular fish feed only. Fish growth is presented aspercentage of weight addition relative to the initial fish weight.

FIG. 6 shows the effect of consuming fish regular food comprisingtransgenic algae overexpressing vacuole targeted-fGH on deformations inshape of koi fish. 398—Regular fish food comprising 4% of fGH expressingalgae. Control—regular fish food only.

FIG. 7 shows the effect of consumption of transgenic algaeoverexpressing fGH on the total weight of goldfish. 398—Regular fishfood comprising 4% of vacuole targeted-fGH expressing algae.Control—regular fish food only.

FIGS. 8A-8C show pictures of brine shrimp (Artemia) fed with wild typePhaeodactylum tricornutum (FIG. 8A), wild type Nannochloris (FIG. 8B)and Phaeodactylum tricornutum expressing vacuole targeted-fish growthhormone (FIG. 8C). Feeds were supplemented as live algae.

FIG. 9 shows the body length of Brine Shrimp fed with wild typeNannochloris (control); with wild type Phaeodactylum tricornutum (w.t.);or with Phaeodactylum tricornutum expressing fGH targeted to the vacuole(398). Feeds were supplemented as live algae.

FIG. 10 shows the effect of food comprising microalgae expressing fGH onthe weight of the shrimp Macrobrachium rosenbergii. 398—Shrimps fed withregular food supplemented with transgenic algae overexpressing fishgrowth hormone targeted to the algae vacuole. Control—shrimps fed withregular food only. Shrimp weight was measured after 6 weeks.

FIGS. 11A-11B show fluorescence imaging of stomachs of Tilapia fish fedwith wild type algae (FIG. 11A) or with vacuole targeted-GFP expressingalgae (construct 527, FIG. 11B). Pictures were taken 4 hours postfeeding under fluorescent light.

FIGS. 12A-12B show fluorescence imaging of intestines of Tilapia fishfed with wild type algae (FIG. 12A) or with vacuole-targeted GFPexpressing algae (FIG. 12B). Pictures were taken 5 hours post feedingunder fluorescent light.

FIGS. 13A-13B show fluorescence imaging of cultures of Brine ShrimpArtemia fed with wild type (FIG. 13A) and vacuole targeted GFPexpressing algae (FIG. 13B). Pictures were taken 4 hours post feedingunder fluorescent light.

FIGS. 14A-14B show pictures of shrimps Macrobrachium rosenbergii fedwith wild type (WT) and vacuole targeted GFP expressing algae takenunder bright (FIG. 14A) and fluorescent (FIG. 14B) light.

FIGS. 15A-15B show pictures of livers isolated from chicks fed with wildtype (WT) and vacuole-targeted GFP expressing algae taken under bright(FIG. 15A) and fluorescent (FIG. 15B) light 6 hours post feeding.

FIG. 16 demonstrates the absorption of the algae-expressed fGH into thefish blood. Tilapia fish were force fed with acuole-GFP expressing algae(construct 527) or with vacuole-fGH expressing algae (construct 398).Level of fGH in the blood was determined using ELISA directedspecifically against the recombinant fGH. Results are shown as mean±SEM.The results are representative of 4 independent experiments.

FIG. 17 demonstrates that an active form of GFP, expressed within thealga vacuole, is absorbed from the fish intestine into its blood in anintact and functional form. Tilapia fish were force fed with vacuole-GFPexpressing algae (construct 527) or with vacuole-fGH expressing algae(construct 398). Level of GFP in the blood was determined usingfluorescence plate reader (515 nm). Results are shown as mean±SD. Theresults are representative of 3 independent experiments.

FIGS. 18A-18B demonstrate that exogenous proteins targeted to the algacell vacuole are protected from degradation in fish gastrointestinaltract. FIG. 18A shows the initial GFP-fluoresces obtained fromvacuole-targeted GFP expressing algae (construct 527); total proteinextract of these algae (GFP) and from vacuole-targeted fGH expressingalgae (construct 398). FIG. 18B shows the fluorescence obtained fromblood samples of Tilapia fish fed with each of the above-described algaeor protein extract. Level of GFP in the blood was determined usingfluorescence plate reader (515 nm). Results are shown as mean±SD.

FIG. 19 demonstrates that exogenous fGH targeted to the algae cellvacuole reach the blood and liver of mice fed with the algae. Mice werefed with vacuole-fGH expressing algae (construct 398) or vacuole-GFPexpressing algae (construct 527). Level of fGH in the liver wasdetermined using ELISA directed specifically against the recombinantfGH. Results are shown as mean±SD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for oraldelivery of biologically active proteins to an organism in need of suchproteins. Particularly, the present invention provides microalgaeexpressing the biologically active protein and edible compositionscomprising same. The present invention demonstrates that the biologicalactivity of the protein is maintained within the consumed algae andfurthermore, that the protein exerts its biological activity in cells ortissues of the organism consuming the transgenic microalgae.

Definitions

The terms “microalga” or “microalgae” is used herein in its broadestscope and refer to unicellular microscopic eukaryotic algae, typicallyfound in freshwater and marine systems. Depending on the species, themicroalgae size can range from a few micrometers (μm) to a few hundredsof micrometers. According to certain currently specific embodiments, theterm refers to marine eukaryotic microalga or microalgae.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of RNA or apolypeptide. A polypeptide can be encoded by a full-length codingsequence or by any part thereof. The term “parts thereof” when used inreference to a gene refers to fragments of that gene. The fragments mayrange in size from a few nucleotides to the entire gene sequence minusone nucleotide. Thus, “a nucleic acid sequence comprising at least apart of a gene” may comprise fragments of the gene or the entire gene.

The term “gene” optionally also encompasses the coding regions of astructural gene and includes sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of about 1 kb on eitherend such that the gene corresponds to the length of the full-lengthmRNA. The sequences which are located 5′ of the coding region and whichare present on the mRNA are referred to as 5′ non-translated sequences.The sequences which are located 3′ or downstream of the coding regionand which are present on the mRNA are referred to as 3′ non-translatedsequences.

The terms “protein”, “protein sequence” and amino acid sequence” areused interchangeably throughout the specification to designate a linearseries of amino acid residues connected one to the other by peptidebonds. The term also encompasses peptides.

The terms “polynucleotide”, “polynucleotide sequence” and “nucleic acidsequence” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA or hybrid thereof, that is single- or double-stranded, linearor branched, and that optionally contains synthetic, non-natural oraltered nucleotide bases. The terms also encompass RNA/DNA hybrids.According to certain currently exemplary embodiments, thepolynucleotides of the present invention are designed based on the aminoacid sequence of the protein of interest employing a codon usage of theparticular microalga species to be transformed.

The terms “expression cassette” and “construct” or “DNA construct” areused herein interchangeably and refer to an artificially assembled orisolated nucleic acid molecule which includes the polynucleotideencoding the protein of interest. The construct may further include amarker gene which in some cases can also encode a protein of interest.The expression cassette further comprising appropriate regulatorysequences operably linked to the polynucleotide encoding the protein ofinterest. It should be appreciated that the inclusion of regulatorysequences in a construct is optional, for example, such sequences maynot be required in situations where the regulatory sequences of a hostcell are to be used.

According to certain embodiments, the organism comprises an expressioncassette comprising operably linked a promoter sequence, apolynucleotide encoding the protein of interest and a terminationsequence.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation.

The terms “promoter element,” “promoter,” or “promoter sequence” as usedherein, refer to a DNA sequence that is located upstream to the 5′ end(i.e. proceeds) the protein coding region of a DNA polymer. The locationof most promoters known in nature precedes the transcribed region. Thepromoter functions as a switch, activating the expression of a gene orpart thereof. If the gene is activated, it is said to be transcribed, orparticipating in transcription. Transcription involves the synthesis ofmRNA from the gene. The promoter, therefore, serves as a transcriptionalregulatory element and also provides a site for initiation oftranscription of the gene into mRNA. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene or partthereof in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofsome variation may have identical promoter activity. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”.

According to the teachings of the present invention, the promoter can bethe organism's native promoter or a heterologous promoter, which may bea constitutive promoter, an induced promoter or a tissue specificpromoter.

Any promoter known in the art to be active in microalgae can be usedaccording to the teachings of the present invention. Non-limitingexamples are fucoxanthin chlorophyll protein A (fcpA); B (fcpB); C(fcpC) and E (fcpE) promoters as well as any light harvesting complex(Lhc) promoter. Non-light harvesting related promoters can also be used,including, but not limited to, the nopaline synthase promoter;poly-adenylation sequences from the Ti plasmid of Agrobacteriumtumefaciens; the promoter region of the tubB2; the PL promoter frombacteriophage λ; the CaMV 35S promoter; the bacterial tφ promoter; theheat shock protein 70A promoter (HSP70A); and a promoter of Rubiscosmall subunit 2 (RBCS2).

As used herein, the term “food” refers to food for human or animalconsumption, including land and aquatic animal.

The term “aquaculture” as used herein refers to aquatic organismcultivated under controlled conditions. An “aquatic organism” is anorganism grown in water, either fresh- or saltwater. Aquatic organisms,include, but are not limited to, fish, e.g., bass, striped bass,tilapia, catfish, sea bream, rainbow trout, zebra fish, red drum,goldfish, Koi fish, Angel fish and carp; crustaceans, e.g., penaeidshrimp, brine shrimp, freshwater and saltwater shrimp, and Artemia; androtifers.

Specific Embodiments for Carrying Out the Invention

The teachings of the present invention are illustrated below with regardto animals, particularly animals grown in aquaculture and model landanimals as non-limiting examples for implementation of at least someaspects of the present invention.

Currently available aquaculture systems are generally classified as openor closed. Open systems are typically created by building a net-pen in abody of water, such as a lake or stream. Closed systems generallyrecirculate the water in a closed tank, the water being pumped from thetank through a treatment cycle and back into the tank.

Aquaculture systems are used to grow aquatic animals such as fish,crustaceans and mollusks, to a size where they are marketable fordifferent uses, primarily as food products but also as ornamentals.According to at least some embodiments the present invention providesimproved food for fish or other aquatic animal. Suitable food forms animportant aspect of aquaculture systems; the teachings of the presentinvention provide means and methods for producing food with enhancedoutcome including improved growth (including enhancing the length and/orweight gain), improved growth pattern (particularly reducing oreliminating body deformation), shortening the time to sexual maturationor another life cycle stage, improved overall health (includingincreasing the survival rate) or combinations thereof.

Oral administration of an edible composition comprising a biologicallyactive protein is of significant economical value in aquaculture as wellas in agriculture, eliminating the need to administer the composition toeach animal individually.

Therapeutic edible compositions are also highly desired for humans, astheir administration does not require professional manpower (requiredfor administration of a therapeutic compound e.g. intravenously) and areeasy to consume thus enhancing the compliance of the patients in takingthe prescribed dose.

According to one aspect, the present invention provides a transgeniceukaryotic microalga comprising an expression cassette comprising atleast one transcribable polynucleotide encoding a biologically activeexogenous protein, wherein the biologically active exogenous protein isexpressed within a subcellular compartment of the microalga cell.

Various algae species can be used according to the teachings of thepresent invention. According to certain embodiments, the alga is marinemicroalga. An exemplary list of marine microalga that can be usedaccording to the teachings of the present invention includes, but is notlimited to, Phaeodactylum tricornutum; Dunaliella spp.; Nannochloropsisspp. including Nannochloropsis oculata, Nannochloropsis salina,Nannochloropsis gaditana; Nannochloris spp., Tetraselmis spp. includingTetraselmis suecica, Tetraselmis chuii; Isochrysis galbana; Pavlovaspp.; Amphiprora hyaline; Chaetoceros muelleri; and Neochlorisoleoabundans. The algae come from and represent a large taxonomicalcross section of species (Table 1).

TABLE 1 Phylogeny of some of the eukaryotic algae Genus Family OrderPhylum Kingdom Phaeodactylum Phaeodactylaceae NaviculalesBacillariophyta Chromalveolata Dunaliella DunaliellaceaeChlamydomonadales Chlorophyta Viridaeplantae Nannochloris CoccomyxaceaeChlorococcales Chlorophyta Viridaeplantae Tetraselmis ChlorodendraceaeChlorodendrales Chlorophyta Viridaeplantae NannochloropsisMonodopsidaceae Eustigmatales Heterokontophyta Chromobiota PavlovaPavlovaceae Pavlovales Haptophyta Chromobiota Isochrysis IsochrysidaceaeIsochrysidales Haptophyta Chromobiota

Phylogeny according to Guiry, M D and Guiry G M. 2013. AlgaeBase.World-wide electronic publication, National University of Ireland,Galway.

According to certain specific embodiments, the transgenic microalga usedaccording to the teachings of the present invention is Phaeodactylumtricornutum. The alga Phaeodactylum tricornutum is a diatomaceousunicellular alga that forms part of phytoplankton and originates fromtemperate climes. This alga is readily amenable to transformation andthe transformed alga growth well in aquaculture. In addition, this algais nontoxic and nonpathogenic, and can be used as a food source foranimals, especially fish and marine invertebrates but also for landanimals.

The primary use of the transgenic microalgae of the present invention isas an edible composition. The exogenous protein expressed in the algalcell should reach the target cell or tissue of the subject consuming thecomposition in its active form, wherein the subject is aquatic or landanimal, including humans. One of the principal obstacles in oraldelivery of a biologically active protein is the susceptibility of theprotein to the environmental conditions throughout the process ofpreparing the oral delivery product and its storage and thereafterwithin the body of the target subject in the gastrointestinal tract.

The present invention now shows that the exogenous protein expressedwithin a subcellular compartment of the microalga preserves its activitywhen consumed by aquatic as well as by terrestrial animals. Withoutwishing to be bound by any specific theory or mechanism of action, theprotein activity may be preserved by the intact alga cell, particularlyby the cell walls, which may act as a form of encapsulation that protectthe protein from the outside harsh environment throughout the growth andprocessing of the algal biomass and furthermore from the environment ofthe gastrointestinal tract of the subject animal consuming the algae.

According to certain embodiments, the subcellular compartment isselected from the group consisting of vacuole, endoplasmic reticulum,Golgi system, lysosome and peroxisome. Each possibility represents aseparate embodiment of the present invention. According to certaincurrently specific embodiments, the exogenous protein is expressedwithin the microalga cell vacuole. Expressing the exogenous proteinwithin the alga chloroplast is explicitly excluded from the presentinvention.

Another problem to be solved in oral delivery of proteins is thepenetration of proteins and peptides through the gastrointestinalepithelial cell membranes of the target animal subject that strictlylimits their penetration. A minimum level of lipophilicity is needed forthe proteins to partition into epithelial cell membranes fortranscellular absorption. Unexpectedly, the present invention now showsthat targeting the polynucleotides to be expressed within the plantvacuole lead to efficient transfer of the expressed, biologically activeprotein into the blood stream of the animal consuming the transgenicmicroalgae. Targeting the protein into the vacuole was advantageous overtargeting to other cell compartments, including chloroplasts. Vacuolesare part of the endomembrane system of a cell; therefore, withoutwishing to be limited by a single hypothesis or mechanism of action,targeting peptides or proteins to the microalga cell vacuole, which ispart of the endomembrane system, may increase absorption through thegastrointestinal tract of the animal once the alga is consumed and itswalls are degraded by the animal subject. Such an increase in absorptionmay be due to increasing the “perceived” lipophilicity of peptide andprotein molecules by the epithelial cell membranes, resulting inefficient absorption through the intestine. In addition, it is alsopossible that providing the protein through the vacuole increasesstorage stability of the protein. Various combinations of the above mayalso play a role. In any case, targeting the protein to the vacuoleclearly increases the functional efficacy of orally administeredproteins, as described an exemplified below in greater detail.

Additionally, exogenous protein expressed by the microalgae can be sodesigned to enhance its uptake by the epithelial cell membranes of theanimal subject consuming the transgenic algae. According to someembodiments, the expression cassette of the present invention furthercomprises a polynucleotide encoding a protein domain that enhances theuptake of the expressed exogenous protein by a xenogeneic cell ortissue.

The particular uptake enhancing domain is selected according to the typeof the xenogeneic cell, which depends on the species of the subjectanimal consuming the transgenic microalgae. According to certainembodiments, the expression cassette further comprises a polynucleotideencoding a cell penetrating peptide (CPP). According to someembodiments, the CPP is selected from the group consisting of, but notlimited to, the trans-activating transcriptional activator (TAT) fromHuman Immunodeficiency virus 1 synthesized according to thePhaeodactylum tricornutum codon usage (SEQ ID NO:9) or part thereof; andthe membrane translocating sequence (MTS) of a fibroblast growth factorsynthesized according to the Phaeodactylum tricornutum codon usage (SEQID NO:7) or part thereof. Each possibility represents a separateembodiment of the present invention.

Proteins having various biological activities can be expressed in themicroalga cell according to the teachings of the present invention.According to certain embodiments, the protein has a therapeutic effecton the subject consuming the transgenic microalga. According to otherembodiments, the protein enhances the growth of the subject consumingthe transgenic microalga. According to yet additional embodiments, theprotein enhances the survival of the subject consuming the transgenicmicroalgae. According to yet additional embodiments, the proteinenhances the reproduction rate of the subject consuming the transgenicmicroalgae.

According to certain exemplary embodiments, the transgenic microalgaexpresses a hormone. According to some embodiments, the hormone isselected from the group consisting of appetite inducing hormone,gonadotropin releasing hormone (spawning hormone) and a growth hormone.According to certain currently specific embodiments the growth hormoneis fish growth hormone.

It is to be understood that the present invention excludes use of thetransgenic microalgae of the present invention as a source of exogenousproteins for vaccines.

Any method for transforming microalgae as is known in the art can beused according to the teachings of the present invention. Transformationmethods include particle bombardment, electroporation, microporation,vortexing cells in the presence of exogenous DNA, acid washed beads andpolyethylene glycol-mediated transformation. Methods and tools fortransformation of eukaryotic algae can be found, for example, inInternational (PCT) Application Publication No. WO 1997/039106.

Typically, to prepare vectors for making the transgenic algae, thepolynucleotide encoding the exogenous protein is first cloned into anexpression vector, a plasmid that can integrate into the algal genome.In such an expression vector, the DNA sequence which encodes theexogenous protein is operatively linked to an expression controlsequence, i.e., a promoter, which directs mRNA synthesis. As describedhereinabove, the promoter can be an endogenous promoter, i.e., apromoter that directs transcription of genes that are normally presentin the algae. According to certain embodiments, the vector furthercomprises a polynucleotide encoding a resistance gene to enableselection of transformed algae. According to certain currently exemplaryembodiments, the vector comprises a polynucleotide encoding a proteinconferring resistance to zeocine and phleomycin.

Culturing conditions of the transformed algae depend on the alga speciesused, as is known to the skilled Artisan and as exemplified hereinbelow.Typically, the algae are grown under conditions that enablephotosynthesis. Since photosynthesis requires sunlight and CO₂ and themicroalgae further require either fresh, brackish or marine water mixedwith the appropriate fertilizers to grow, microalgae can be cultivatedin, for example, open ponds and lakes. However, the open systems aremore vulnerable to contamination than a closed system, and furthermore,genetically modified microalgae grown in open aqueous reservoirs may betaken as hazardous to the environments. In addition, in open systemsthere is less control over water temperature, CO₂ concentration, andlighting conditions. The growing season is largely dependent on locationand, aside from tropical areas, is limited to the warmer months of theyear. An open system, however, is cheaper to set up and/or maintain thana closed system.

Another approach to growing the microalgae is thus to use a semi-closedsystem, such as covering the pond or pool with a structure, for example,a “greenhouse-type” structure. While this can result in a smallersystem, it addresses many of the problems associated with an opensystem. The advantages of a semi-closed system are that it can allow forthe desired microalgae to be dominant over an invading organism byallowing the microalgae of interest to out-compete the invading organismfor nutrients required for its growth, and it can extend the growingseason. For example, if the system is heated or cooled, the microalgaecan grow year round.

Alternatively, the microalgae can be grown in closed structures suchasphotobioreactors, where the environment is under stricter control thanin open systems or semiclosed systems. A photobioreactor is a bioreactorwhich incorporates some type of light source to provide photonic energyinput into the reactor. The term photobioreactor can refer to a systemclosed to the environment and having no direct exchange of gases andcontaminants with the environment. A photobioreactor can be described asan enclosed, illuminated culture vessel designed for controlled biomassproduction of phototrophic liquid cell suspension cultures. Examples ofphotobioreactors include, for example, glass containers, plastic/glasstubes, tanks, plastic sleeves, and bags. Examples of light sources thatcan be used to provide the energy required to sustain photosynthesisinclude, for example, fluorescent bulbs, LEDs, and natural sunlight.Because these systems are closed everything that the organism needs togrow (for example, carbon dioxide, nutrients, water, and light) must beintroduced into the bioreactor. Photobioreactors, despite the costs toset up and maintain them, have several advantages over open systems,they can, for example, prevent or minimize contamination, offer bettercontrol over the culture conditions (for example, pH, light, carbondioxide, and temperature), prevent water evaporation, lower carbondioxide losses due to degassing, and permit higher cell concentrations.On the other hand, certain requirements of photobioreactors, such ascooling, mixing, control of oxygen accumulation and bio-fouling, makethese systems more expensive to build and operate than open systems orsemi-closed systems. Photobioreactors can be set up to be continuallyharvested (as is with the majority of the larger volume cultivationsystems), or harvested one batch at a time (for example, as withpolyethylene bag cultivation). A batch photobioreactor is set up with,for example, nutrients, microalgae, and water, and the microalgae isallowed to grow until the batch is harvested. A continuousphotobioreactor can be harvested, for example, either continually,daily, or at fixed time intervals.

CO₂ can be delivered to any of the systems described herein, forexample, by bubbling in CO₂ from under the surface of the liquidcontaining the microalgae. Also, sparges can be used to inject CO₂ intothe liquid. Spargers are, for example, porous disc or tube assembliesthat are also referred to as Bubblers, Carbonators, Aerators, PorousStones and Diffusers.

Nutrients that can be used in the systems described herein include, forexample, nitrogen (in the form of NO₃ ⁻ or NH₄, phosphorus, and tracemetals (Fe, Mg, K, Ca, Co, Cu, Mn, Mo, Zn, V, and B). The nutrients cancome, for example, in a solid form or in a liquid form. If the nutrientsare in a solid form they can be mixed with, for example, fresh or saltwater prior to being delivered to the liquid containing the microalgae,or prior to being delivered to a photobioreactor.

The microalgae can be grown in large scale cultures, where large scalecultures refers to growth of cultures in volumes of greater than about 6liters, or greater than about 10 liters, or greater than about 20liters. Large scale growth can also be growth of cultures in volumes of50 liters or more, 100 liters or more, or 200 liters and up.

Optimal growth temperature is typically about 20° C. to about 25° C.,however it is species dependent. According to certain embodimentsmicroalgae cell reach a density of 10⁵ to 10⁸/ml before harvesting.

Post-harvest processing of some sort may be used to prepare the materialfor oral consumption or as a food composition. Conventional processestypically include at least partial separation of the algal biomass fromthe liquid culture in which the algae were grown. Optionally, the algalbiomass can be homogenized and/or dried to form pellets of varioussizes, depending on the target subject and mode of application. Othermodes of preparation include spray drying, fluid bed drying, or evenproviding the material as a liquid suspension.

The harvested transgenic microalgae of the present invention can beadministered per se, can be formulated into an edible compositionfurther comprising edible diluents, excipients or carriers. Themicroalgae or the composition comprising same can be further used asfood additive. According to some embodiments, the edible composition isan animal food composition. According to certain currently specificembodiments, the animal food composition if for feeding aquatic and landanimals. According to yet other embodiments, the edible composition isfor human consumption.

According to a further aspect the present invention provides a methodfor improving at least one of an animal growth rate, growth pattern,reproductive health status, survival or any combination thereof,comprising administering to the animal transgenic microalgae of thepresent invention or a composition comprising same, thereby improvingthe growth rate and/or the growth pattern and/or the survival and/or thereproductive health status of said animal.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

These Examples relate to specific implementations of at least someaspects of embodiments of the present invention. The Examples areillustrative only and are not intended to be limiting in any way.

Materials & Methods

Synthesis of Salmon Growth Hormone Gene

The amino acid sequence of the Salmon growth hormone (accession No.AAT02409) was used as the basis for the synthesis of a polynucleotideencoding a mature Salmon growth hormone (SEQ ID NO:12, designated herein“fish growth hormone” or “fGH”). The polynucleotide synthesis wasperformed using the Phaeodactylum tricornutum codon usage of “EntelechonGmbH”, thereby forming a novel polynucleotide sequence shown in SEQ IDNO:1. This novel sequence was not previously disclosed, as this codonusage has not yet been used for this protein. Furthermore, this specificsequence was designed to be more efficiently expressed by Phaeodactylumtricornutum, as discussed in greater detail below.

Construction of the Salmon Growth Hormone Gene Targeted to ER

The fGH growth hormone gene was fused at its 5′ end to a polynucleotideencoding Bip endoplasmic reticulum leader sequence (Kilian O and KrothP. 2005. The Plant Journal: 41:175-183) (nucleic acids: SEQ ID NO:16;amino acids: SEQ ID NO:2) to produce the Bip-fGH (nucleic acids: SEQ IDNO: 17; amino acids: SEQ ID NO:3), according to the following:

The Bip leader sequence was amplified from Phaeodactylum tricornutumgenomic DNA using the following primers:

Forward BiP: (SEQ ID NO: 36) GGAATTCATGATGTTCATGAGAATTGCReverse Bip-fGH-V2 (SEQ ID NO: 37) ACGCTGGTTTTCAATCACGGTACCCATCTT.

The Bip leader sequence product was amplified using the followingprimers:

Forward BiP (SEQ ID NO: 38) GGAATTCATGATGTTCATGAGAATTGC.Reverse Bip-fGH-V2 (SEQ ID NO: 39) ACGCTGGTTTTCAATCACGGTACCCATCTT.

The fGH was amplified using the following primers:

Forward Bip-fGH-V2 (SEQ ID NO: 40) AAGATGGGTACCGTGATTGAAAACCAGCGT.Reverse fGH BglII (SEQ ID NO: 41) GAGATCTGAGGGTGCAGTTGG.

The Bip leader sequence was fused to fGH by a third PCR, using theamplified PCR products (Bip, fGH), and the mentioned primers for Bip+Revf BgIII, resulting in the construct designated 356 having the nucleicacid sequence set forth in SEQ ID NO:17 and the amino acid sequence setforth in SEQ ID NO:3.

Construction of the Salmon Growth Hormone Gene Targeted to Vacuole

The fGH encoding polynucleotide was fused to a vacuole leader sequence(nucleic acids: SEQ ID NO:18; amino acids: SEQ ID NO:4) at its 5′ and toan HA tag (nucleic acids: SEQ ID NO:47; amino acids: SEQ ID NO:5) at its3′ to produce the vacuole-fGH-HA polynucleotide (nucleic acids: SEQ IDNO:19; amino acids: SEQ ID NO:6), according to the following:

The synthetic fGH was amplified using the following primers:

Forward fGH EcoRI (SEQ ID NO: 42) GGAATTCATGGGCCAAGTCTTTCTCTTGReverse fGH BglII (SEQ ID NO: 41) GAGATCTGAGGGTGCAGTTGG.

The product was ligated to a pPhaT1-HA plasmid using EcoRI, BgIII.

The fGH-HA template was amplified using the following primers:

Forward fGH BamHI: (SEQ ID NO: 43) GGATCCATTGAAAACCAGCGTTTGTTCAAC.Reverse Hind HA: (SEQ ID NO: 44)AAGCTTTTACTGGGCGGCGTAGTCCGGGACGTCGTAGGGGTA.

The PCR product was cloned into pPhaT1 by BamHI and HindIII.

The vacuole leader sequence was amplified from Phaeodactylum tricornutumcDNA using the following primers:

EcoR1-Vac54681: (SEQ ID NO: 45) ATGAATTCATGTCGATTCGTCTCT.BamH1-Vac54681: (SEQ ID NO: 46) ATGGATCCAGTTTGGGCAGTTGCC.

The fGH-HA and the vacuole leader sequence were ligated with EcoRI andBamHI, resulting in the construct designated 398, having the nucleicacid sequence set forth in SEQ ID NO: 19 encoding the polypeptide havingthe amino acids sequence set forth in SEQ ID NO:6.

Construction of the GFP-Encoding Polynucleotide Targeted to Vacuole

In the construct described above, the polynucleotide encoding the fishgrowth hormone was replaced with a polynucleotide encoding GFP(accession P42212, having the nucleic acid sequence set forth in SEQ IDNO:29 and encoding a protein having the amino acid sequence set forth inSEQ ID NO:28) by BamHI and HindIII, leading to a vacuole targeted GFP(SEQ ID NO:30, amino acid sequence; SEQ ID NO:31, nucleic acidsequence—prepared with Phaeodactylum tricornutum codon usage), resultingin a construct designated 527, having the nucleic acid sequence setforth in SEQ ID NO:31 and the amino acid sequence set forth in SEQ IDNO:30.

Construction of the Vacuole-fGH-MTS-HA

A membrane translocating sequence (MTS) from the fibroblast growthfactor was synthesized according to the Phaeodactylum tricornutum codonusage by Biomatik (SEQ ID NO:7). The translocating sequence was ligatedto the vacuole-fGH-HA at the 3′ of fGH using BgIII, to produce theconstruct encoding vacuole-fGH-MTS-HA having the amino acid sequence setforth in SEQ ID NO:8.

Construction of the Vacuole-GFP-MTS

A membrane translocating sequence (MTS) from the fibroblast growthfactor was synthesized according to the Phaeodactylum tricornutum codonusage by Biomatik. The translocating sequence was ligated to thevacuole-GFP at the 3′ of GFP using HindIII, to produce thevacuole-GFP-MTS construct (nucleic acid sequence: SEQ ID NO:33; aminoacid sequence SEQ ID NO: 32).

Construction of the Vacuole-fGH-TAT-HA

A trans-activating transcriptional activator (TAT) from HumanImmunodeficiency virus 1 (HIV-1) was synthesized according to thePhaeodactylum tricornutum codon usage by Biomatik (SEQ ID NO:9). Thedomain was fused by BgIII in tandem of two repeats to the vacuole-fGH-HAat the 3′ of fGH to produce the vacuole-fGH-TAT-HA construct (SEQ IDNO:10).

Construction of the Vacuole-GFP-TAT

A trans-activating transcriptional activator (TAT) from HumanImmunodeficiency virus 1 (HIV-1) was synthesized according to thePhaeodactylum tricornutum codon usage by Biomatik. The gene was ligatedto the vacuole-GFP at the 3′ of GFP using HindIII, to produce thevacuole-GFP-TAT construct having the nucleic acid sequence set forth inSEQ ID NO:35, encoding the vacuole-GFP-TAT protein having the amino acidsequence set forth in SEQ ID NO:34.

Cloning Constructs into an Algae Expression Vector

The various polynucleotides and constructs of the invention were furthercloned under the control of the fcpA promoter and fcpA terminator in theplasmid pPHAT1 (accession number AF219942) (SEQ ID NO:11) according toApt et al. (1996. Mol. Gen Genet. 252:572-579). The fcpA promoter is theonly one that is currently known to be operative in Phaeodactylumtricornutum. However, it is to be explicitly understood that otherpromoters can be used in Phaeodactylum tricornutum as well as in otheralgae.

The vector contained:

-   -   An fcpA (fucoxanthin chlorophyll protein A) promoter, under        which the gene of interest is cloned.    -   MCP—Multiple cloning site    -   An fcpB (fucoxanthin chlorophyll protein B) promoter, which        controls the sh ble gene from Streptoalloteichus hindustanus,        which encodes a protein that confers zeocine and phleomycin        resistance.    -   fcpA terminators, which appear after the gene of interest and        after the zeocine resistance gene.    -   Ampicillin resistant gene    -   Origin of replication from Escherichia coli.

FIG. 1 shows the complete pPhaT1 expression vector.

The fGH encoding polynucleotide (SEQ ID NO:1) was cloned under the fcpApromoter and fcpA terminator. The plasmid contained the selectablemarker, Bleomycine, under the control of the fcpB promoter and fcpAterminator.

Cloning and Molecular Techniques

PCR reactions were done using Phusion Polymerase Cat.# FZ-F-530SFinnzymes (Zotal) or REDTaq ready mix PCR reaction mix Cat.#R2523-100RXN Sigma. PCR reactions were cleaned using Wizard® SV Gel andPCR Clean-Up System (Cat. No. A9281, Promega).

Ligations were performed using DNA Ligation Kit (Mighty Mix)-Takara Cat.No. 6023 (Ornat) or T4 DNA ligase MO202T NEB (Eldan). Blunting of 5′ or3′ overhangs was performed with T₄ DNA polymerase: (Fermentas #EP0061).

DNA Midi preps were performed using Pure Yield™ Plasmid Midiprep SystemA2492. PROMEGA and DNA minipreps were performed using AccuPrep PlasmidMini Extraction Kit-BIONEER K-3030. DNA genomic isolations wereperformed according to Fawley & Fawley (Fawley M W and Fawley K P. 2004.J Phycol 40: 223-225). All kits and enzymes were treated according tothe manufacturer's instructions.

Algae Culturing and Harvesting

Algae culturing and harvesting was done as described in U.S. PatentApplication Publication No. 2011/0081706 to the Applicant of the presentinvention. Briefly, algae were cultured in filtered sea water enrichedwith F/2 nutrient for growing diatoms (modified from Andersen R et al.2005. Recipes for freshwater and seawater media. In: Algal CulturingTechniques (R. A. Andersen, eds), pp. 429-538. Elsevier, Amsterdam). F/2was added every 72 h at a dosage of 1:1000 to the final culture volume.A constant temperature regime was maintained at 21° C. Light: dark wasset at 16:8 hours at a light intensity of 100 μmol photons per m² s¹.CO₂ was mixed with air and delivered to the cultures at controlled ratiovia the aeration systems. Algae were harvested for experiment near theirmaximal culture densities. To help flocculation of the algae calciumhydroxide was added to the culture as a fine suspension of particles inwater containing 0.15 g/ml Ca(OH)₂, and the culture was then filtered orcentrifuged. The resulting algae sediment was lyophilized.

Algae Transformation

I. Transformation by Particle Bombardment

Fresh algal culture were grown to mid exponential phase (2-5*10⁶cells/ml) in artificial sea water (ASW) F/2 media as described above. 24hours prior to bombardment cells were harvested, washed twice with freshASW+F/2 and resuspended in 1/10 of the original cell volume in ASW+F/2.0.5 ml of the cell suspension is spotted onto the center of a 55 mmPetri dish containing solidified ASW+F/2 media. Plates are left to dryunder normal growth conditions. Bombardment was carried out using a PDS1000/He biolistic transformation system according to the manufacturer'sinstructions (BioRad Laboratories Inc., Hercules, Calif. USA) using M17tungsten powder (BioRad Laboratories Inc.) for cells larger than 2microns in diameter, and tungsten powder comprised of particles smallerthan 0.6 microns (FW06, Canada Fujian Jinxin Powder Metallurgy Co.,Markham, ON, Canada) for smaller cells. The tungsten was coated withlinear DNA. 1100 or 1350 psi rupture discs were used. All disposableswere purchased from BioRad Laboratories Inc. After bombardment theplates were incubated under normal growth conditions for 24 hours afterwhich the cells were plated onto selective solid media and incubatedunder normal growth conditions until single colonies appeared.

II. Transformation by Electroporation

Algal cultures were grown to mid exponential phase in artificialseawater (ASW)+F/2 media as described above. Cells were then harvestedand washed twice with fresh media. After re-suspending the cells in 1/50of the original volume, protoplasts were prepared by adding an equalvolume of 4% hemicellulase (Sigma) and 2% Driselase (Sigma) in ASW andwere incubated at 37° C. for 4 hours. Protoplast formation was tested byCalcofluor white non-staining. Protoplasts were washed twice with ASWcontaining 0.6M D-mannitol and 0.6M D-sorbital and resuspended in thesame media, after which DNA was added (10 μg linear DNA for each 100 μlprotoplasts). Protoplasts were transferred to cold electroporationcuvettes and incubated on ice for 7 minutes, then pulsed in an ECM830electroporation apparatus (BTX, Harvard Apparatus, Holliston, Mass.,USA). A variety of pulses is usually applied, ranging from 1000 to 1500volts, 10-20 msec per pulse. Each cuvette was pulsed 5-10 times.Immediately after pulsing the cuvettes were placed on ice for 5 minutesand then the protoplasts were added to 250 μl of fresh growth media(non-selective). After incubating the protoplasts for 24 hours in lowlight at 25° C. the cells were plated onto selective solid media andincubated under normal growth conditions until single colonies appeared.

III. Transformation by Microporation

A fresh algal culture was grown to mid exponential phase in ASW+F/2media. A 10 ml sample of the culture was harvested, washed twice withDulbecco's phosphate buffered saline (DPBS, Gibco, Invitrogen, Carslbad,Calif., USA) and resuspended in 250 μl of buffer R (supplied by DigitalBio, NanoEnTek Inc., Seoul, Korea, the producer of the microporationapparatus and kit). After adding 8 μg linear DNA to every 100 μl cells,the cells were pulsed. A variety of pulses is typically needed,depending on the type of cells, ranging from 700 to 1700 volts, 10-40msec pulse length; each sample was pulsed 1-5 times. Immediately afterpulsing, the cells were transferred to 200 μl fresh culture media(non-selective). After incubating for 24 hours in low light at 25° C.,the cells were plated onto selective solid media and incubated undernormal culture conditions until single colonies appeared.

Protein Extraction

10 ml cells at 5×10⁶ cell/ml were harvested and resuspended in 500 μlextraction buffer (50 mM Tris pH=7.0; 1 mM EDTA; 100 mM NaCl; 0.5%NP-40; and protease inhibitor (Sigma cat# P9599). Then 100 μl of glassbeads (425-600 μTTI, Sigma) were added and cells were broken in a beadbeater (MP FastPrep-24, MP Biomedicals, Solon, Ohio, USA) for 20 sec.The tube content was centrifuged for 15 min, 13000×g, at 4° C. Thesupernatant was removed to new vial for quantification and Western blotanalysis.

Protein Separation by SDS-PAGE and Western Analysis

Extracted proteins were separated on a 4-20% gradient SDS-PAGE (Gebagels 4-20% Bio-lab, 10GG0420-8), at 100V for 1 h. Following incubationof 1 h in blocking buffer (5% skim milk, Difco), proteins were eitherstained by Coomassie (Sigma) or blotted onto PVDF (Millipore, Billerica,Mass., USA) membranes for 1 h at 100 volts in transfer buffer (25 mMTris, 192 mM glycine and 20% methanol). The proteins were detectedeither with an anti HA (Biotest MMS-101P-500) or the salmon growthhormone (GroPep: PAN1) antibodies, diluted to a ratio of 1:1000 in theblocking buffer. Mouse (for the HA antibody) or rabbit (for the Salmongrowth hormone) horseradish peroxidases secondary antibodies (Millipore,Billerica, Mass., USA), at 1:10000 dilution in the blocking buffer wereused. Detection was carried out using the EZ-ECL kit (Bio Ind. Promega:20-500-120) according to manufacture instructions.

ELISA Analysis

ELISA plate (microlon, Greiner) was coated in carbonate/bicarbonate pH9.6 with monoclonal anti HA Antibody (Sigma Aldrich) overnight at 4° C.Next, the plate was washed with PBST (0.05% Tween), and blocked with 1%BSA (Sigma Aldrich) in PBS for 4 hours at room temperature (RT). Serumsamples were serially diluted in ELISA coating buffer and loaded ontothe plate. After an overnight incubation with the serum samples, theplate was washed with PBST and incubated with anti HA-biotin (Roche) for1 hour at room temperature. Following additional washing steps,horseradish peroxidase (HRP) conjugated sterptavidin was added and theplate was incubated for 1 hour at room temperature (RT). The plate waswashed with PBST and tetramethyl-benzidine (TMB) substrate was added tothe plate. Once sufficient color was developed, the reaction was stoppedwith 0.16M sulfuric acid. The absorbance in each well was measured at450 nm using Enspire 2300 multilabel reader (PerkinElmer).

Fish Maintenance and Feeding

Groups of 10 Tilapia fish, weighing 70-100 grams were maintained in 100liter aerated tanks at a temperature of 26-28° C. The photoperiod was 12h light: 12 h dark. All fish were acclimated in the tanks for a weekbefore the treatment. Oral administration with algae suspensions wasconducted on fish lightly anesthetized with 100 ppm of clove bud extract(Roth). Polyethylene tube (length 6-8 cm, i.d. 3 mm) attached to aninjecting syringe was used for oral administration (gavage feeding) ofthe different algal suspensions.

Feeding Trials

Lyophilized transgenic algae expressing the Salmon growth hormone wereadded in a final concentration of 1-4% to the regular fish feed. Fishreceived with feed at 10%-15% of their total body weight. The transgenicalgae and the regular fish feed were used to feed the ornamental fish,Koi, Scalare and Goldfish Shubunkin for 6 weeks. Each trial wasmonitored for temperature, pH, ammonia, nitrite levels etc. At the endof each experiment fish total growth, morphological abnormalities andsurvival rates were analyzed.

Example 1 Algae Expressing Fish (Salmon) Growth Hormone

Transgenic algae of the species Phaeodactylum tricornutum, harboring theSalmon growth hormone encoding polynucleotide targeted to the ER(designated as construct 356) or to the vacuole (designated as construct398) were cultured and analyzed for the expression of the transgenicprotein. An equal amount of total soluble protein (20^(˜) g) wasextracted from the transgenic algae for Western blot analysis. Detectionwas made using anti Salmon growth hormone antibody as shown in FIG. 2.

The algae line expressing ER-targeted Salmon growth hormone has shownhigher expression levels of the transgenic protein compared to the algaeline expressing the vacuole-targeted growth hormone.

Example 2 Angel Fish Feeding Trial

Ornamental Angel Fish (Scalare), were fed during 6 weeks with fish foodsupplemented with transgenic Phaeodactylum tricornutum over-expressingfish growth hormone targeted to the ER or to the vacuole (constructs356, 398, respectively), or with the regular, non-transgenic fish food(control). The algae supplement was added at 4% into the regular fishfood. Fish growth was followed within tanks as independent repeats (n=5)containing 20 fish each. FIG. 3 shows the total growth performance ofthe Angel fish treated with the transgenic algae (mixed within the fishfood), compared to fish fed with regular fish food.

Fish fed with food containing the transgenic algae harboring construct398 (in which the growth hormone was targeted to the vacuole) gavehigher total fish biomass (˜15%), compared to fish fed with the regularfood (control), whereas fish fed with food containing the transgenicalgae harboring construct 356 (in which the growth hormone was targetedto the ER) did not grow significantly better compared to the control.

In a parallel experiment, also conducted with Angel fish, feeding thefish with regular food containing algae expressing fGH targeted to thevacuole (construct 398) resulted in an increase in the number of fishreaching over 2 gr compared to fish fed with regular fish food (FIG. 4).From a marketing perspective this result implies that supplementation ofregular fish food with algae expressing fGH targeted to the vacuoleincreases the number of fish which are suitable for marketing, withoutincreasing the number of starting fish in the population or otherwisenecessarily adjusting any aspect of the starting fish population.

Another experiment conducted for 8 weeks with Angel fish in 5 repeats of25 fish each, resulted in a significant biomass increase of fishconsuming food supplemented with 4% algae expressing fGH (fGH expressiontargeted to the vacuole, construct 398) compared to fish fed withregular fish food (FIG. 5).

These results imply that the subcellular targeting of fGH to the vacuoleof the alga cell results in better functional efficacy of the protein,even though the expression level was shown to be lower than theexpression level of fGH targeted to cell ER.

Example 3 Koi Fish Feeding Trial

Post larva koi fish (4 independent repeats each including 600 post larvakoi fish) were fed either with the regular fish food or with regularfish food supplemented with 4% algae expressing fGH targeted to thevacuole (construct 398). The feeding experiment was performed over 8weeks. At the end of the experiment, fish were screened for bodydeformation (as defined by Jha P et. al. 2006. Journal of AppliedIchthyology; 23 (1) 87-92). Body deformation includes but is not limitedto any morphological irregularity, any type of body asymmetry, irregularbody shape, irregular fin shape, irregular tail shape; irregularbody/fin area, length or width ratios; irregular body/tail area, lengthor width ratios; irregular tail/fin area, length or width ratios; orirregularities in any body part. FIG. 6 shows the percentage of normaland deformed shape of fish fed with regular fish food and fish fed withregular food containing 4% of vacuole-targeted fGH expressing algae(construct 398).

As is apparent from FIG. 6, the population of fish receiving regularfish food exhibited 45% deformed fish, while the population of fish thatreceived fish food supplemented with fGH expressing algae (construct398) exhibited only 38% deformed fish.

Example 4 Goldfish Feeding Trial

The post larva ornamental Goldfish (Shubunkin) were fed over 6 weekswith fish food supplemented with transgenic Phaeodactylum tricornutumoverexpressing fish growth hormone targeted to the vacuole (construct398), or with regular fish food (control). Algae supplement was added at4% to the regular fish food. Treatments were tested in 6 independentrepeats of 40 fish each. FIG. 7 shows that the total weight of goldfishfed with transgenic algae was higher by approximately ˜15%, compared tofish fed with regular food.

In addition to the growth performance, the survival of fingerlingsduring the experiment was approximately 64% at the control tanks. Thesurvival rate was elevated to approximately 80% within tanks in whichthe fish were fed with food supplemented with fGH expressing algae(construct 398), implying that the fGH expressing algae contributes tofish survival in addition to its growth enhancement effect.

Example 5 Artemia Feeding Trial

Brine shrimps (Artemia) were fed for 12 days with wild typePhaeodactylum tricornutum, wild type Nannochloris or with fish growthhormone (fGH) expressing Phaeodactylum tricornutum (construct 398).Artemia fed with fGH expressing Phaeodactylum tricornutum weresignificantly larger (by about 40%) and the females reached sexualmaturation 3 days earlier, when compared to Artemia fed with wild typeNannochloris or wild type Phaeodactylum tricornutum respectively (FIGS.8 and 9).

Specifically, FIG. 8 shows the results after brine shrimp were fed withwild type Phaeodactylum tricornutum (FIG. 8A), wild type Nannochloris(FIG. 8B) or with Phaeodactylum tricornutum expressing fish growthhormone. Pictures were taken after 12 days.

FIG. 9 shows the measured average body length of fish receiving thedifferent algae as the sole food. For the determination of body length,the body length of 26 randomly selected Brine Shrimp fed with wild typeNannochloris (control), Wild type Phaeodactylum tricornutum (wt) or fishgrowth hormone expressing Phaeodactylum tricornutum (construct 398) wasmeasured after 12 days. Brine fish fed with fGH expressing algae werefound to be significantly longer. Taken together, these results suggestthat using algae expressing fish growth hormone targeted to the algalvacuole as food or food additive in Brine Shrimps and crustaceansenhance their growth, particularly with regard to body length, andsexual maturation.

Example 6 Macrobrachium rosenbergii Feeding Trial

The fresh water shrimp Macrobrachium rosenbergii, PL 10, were fed over 6weeks with regular food (control), or with regular food supplementedwith transgenic Phaeodactylum tricornutum overexpressing fish growthhormone targeted to the algae vacuole (construct 398). Algae supplementwas added at 8% to the regular food. FIG. 10 shows that the total weightof Macrobrachium rosenbergii treated with transgenic algae wassignificantly higher by 21%, compared to shrimps fed with regular food,implying that the fGH expressing algae contributes to the shrimpenhanced growth.

Example 7 GFP Absorption by Fish

1 week starved Tilapia fish at a size of 50-100 grams were fed with fishfood mixed with wild-type Phaeodactylum tricornutum or withPhaeodactylum tricornutum expressing GFP targeted to the vacuole at 1:1fish food to algae ratio. Tilapia stomachs and intestines were taken out1-4 hours post feeding and were analyzed under fluorescence binocular.FIG. 11 clearly shows fluorescence of the expressed exogenous GFP in thestomachs of the Tilapia four hours post feeding, implying that thefluorescent protein was not degraded in the acidic environment of thestomach. Furthermore, clear fluorescence of the protein was alsoobserved in the intestine (FIG. 12). In summary, these results show thatthe protein is not degraded in the acidic environment of the stomach; itis delivered from the microalgae into the fish intestine; andfurthermore, the protein keeps its biological function under theseconditions.

Example 8 GFP Absorption by Shrimp

The Brine shrimp Artemia were fed with wild type algae (Phaeodactylumtricornutum) alone or with algae expressing GFP targeted to the vacuole(vacuole-GFP expressing algae) alone for 3 days post hatching. 8000Artemia grown in 1× ASW medium were fed with 1.5*10⁹ algae. 4 hours postfeeding, Artemia were carefully washed and analyzed under fluorescentlight. FIG. 13 demonstrates that the GFP protein expressed in algae islocated within the digestive system of the Artemia in its intact andbiological form (FIG. 13B). These results again demonstrate that themicroalgae system disclosed in the present invention is highly suitablefor oral delivery of proteins, which are delivered from the microalgaeand into the recipient digestive system in their intact and biologicallyactive form.

In still additional experiment, starved fresh water shrimps,Macrobrachium rosenbergii, PL10, were fed with pellets composed ofregular food mixed with powder of wild-type algae (Phaeodactylumtricornutum) or with powder of vacuole-GFP expressing algae in food toalgae ratio of 1:1. Shrimps were analyzed under fluorescent light 4 hpost feeding. FIG. 14 demonstrates that the GFP protein is located inthe hepatopancrease gland of the shrimp, implying oral delivery of theGFP protein in this animal. Again the protein was delivered intact andactive.

Example 9 GFP Absorption by Chickens

Two weeks old chickens were force fed each with 100 mg of powder ofwild-type algae (Phaeodactylum tricornutum) or with algae expressing GFPtargeted to the vacuole suspended in 5 ml of 0.5×artificial sea water(ASW). Chickens were sacrificed 2, 4, 6 and 24 h post feeding and liverswere taken out and analyzed under fluorescent light. FIG. 15demonstrates that the GFP protein was absorbed into the chick's liver, 6h post feeding, in its intact and functional form implying that the GFPprotein passed through the acidic digestive tract, followed byabsorption through the intestine wall into the blood, circulating to theliver. The result demonstrates the full path of protein oral delivery tochickens.

Example 10 Oral Delivery of Proteins to Fish Blood

Tilapia fish were fed with algae (Phaeodactylum tricornutum) expressingfish growth hormone or with algae expressing GFP (both targeted to thealgae vacuole; vacuole-fGH expressing and vacuole-GFP expressing algae,respectively). 800 mg of algal powder was suspended in 30 ml of 0.5×ASW.Each fish was force-fed with 2 ml algal suspension. Blood samples weretaken from the caudal vein using sterile syringes and tubes. Fiftymicroliters (μl) of each of the blood samples were allocated for directfluorescence analysis for the activity of GFP in a fluorescence platereader and the rest of the samples were left to stand for 15 minutes atroom temperature, and after an overnight period at 4° C., sera wereseparated by centrifugation at 250 g for 10 minutes at 4° C., and storedin sterile tubes at −20° C. for ELISA analysis.

Absorption of fGH in Tilapia Fish

Tilapia fish were force-fed with vacuole-fGH (construct 398) orvacuole-GFP expressing algae (construct 527). Blood samples were taken 1h post feeding for ELISA analysis. The presence of the fGH protein wasconfirmed by detecting the expression of the HA domain, which is fusedto the fGH using anti-HA antibody (see Materials and Methodshereinabove). The blood samples taken from fish fed with vacuole-fGHexpressing algae, reacted positively with the anti-HA antibody, whileblood samples taken from fish fed with vacuole-GFP expressing algae gavenon-significant signal (FIG. 16). The ELISA results provide support forthe presence of fGH protein in the blood, showing that proteinsexpressed in the algae reach the blood of organisms orally consuming thealgae.

Absorption and Activity of GFP in Tilapia Fish

Tilapia fish (n=5) were force-fed with vacuole-GFP expressing algae(construct 527) or with vacuole-fGH expressing algae (construct 398).Blood sample were collected one hour post feeding and analyzed underfluorescence light (excitation 480 nm, emission 515 nm) using Enspire2300 multi-label reader. Blood samples collecting from fish def withvacuole-GFP expressing algae exhibited fluorescence. In contrast, bloodsamples of fish fed with vacuole-fGH expressing algae (construct 398)gave non-significant signal (FIG. 17). The results indicate that GFP wasabsorbed from the fish intestine into its blood in an intact andfunctional form.

Example 11 Vacuole-Targeted Exogenous Proteins are Protected in theGastrointestinal Tract

Vacuole-GFP and vacuole-fGH expressing algae (harboring construct 527and 398, respectively) and total protein extracted from the vacuole-GFPexpressing algae line were measured for GFP fluorescence before beingadministered to the fish by force feeding (FIG. 18A) and in blood sampletaken from the fish 1 h post application (FIG. 18B). Fluorescence wasmeasured using Enspire 2300 multi-label reader (excitation 480 nm,emission 515 nm). As can be seen, only the blood samples taken from fishfed with GFP expressing algae were fluorescent, while blood samplestaken from fish fed with isolated proteins extracted from thevacuole-GFP expressing algae gave no signal or only a background signalas observed in blood samples taken from fish fed with vacuole-fGHexpressing algae. The results clearly demonstrate that the algae cellprotects the GFP protein and enables its delivery to the blood of theorganism consuming the algae in its intact and functional form. Incontrast, oral administration of naked fluorescent protein (GFP) leadsto abolishment of its fluorescence, probably due to its degradation inthe gastro-intestinal tract.

Example 12 Oral Delivery of Proteins to Mice

Delivery of fGH into Mice Liver

12 weeks old balb-C male mice were starved for 12 hours prior to theexperiment. Mice were lightly anesthetized using Isoflurane, and fedwith 1.5 ml of vacuole-fGH expressing algae (construct 398) orvacuole-GFP expressing algae (construct 527) by gavage-feeding.

Two hours post algal administration mice were euthanized by overdose ofisoflurane. Livers were removed and frozen in liquid nitrogen. Totalprotein was extracted from the livers, followed by ELISA analysisdirected towards the HA tag, recognizing specifically the recombinantfGH, expressed in the algae. The ELISA results, shown in FIG. 19,demonstrate that the recombinant fGH was detectable in the mice livers,indicating its oral pass through the mice digestive tract into the bloodand the liver.

Absorption of GFP to the Blood

Mice were treated as above, and blood samples were taken from the heart2 hours post feeding. The blood samples were then centrifuged and theplasma was analyzed under fluorescent light (excitation 480 nm, emission515 nm). Blood samples of mice fed with vacuole-GFP expressing algaeshowed significantly higher fluorescence compared to blood samples ofmice fed with vacuole-fGH expressing algae, demonstrating the passage ofthe GFP from the digestive tract into the blood in an intact andfunctional form.

Example 13 Enhanced Protein Absorption by Fish and Mice

CPPs are short peptides that facilitate cellular uptake of variousmolecular cargos. Examples of CPPs include the trans-activatingtranscriptional activator (TAT) from Human Immunodeficiency virus 1(HIV-1) and the membrane translocating sequence (MTS) from a fibroblastgrowth factor. The constructs comprising the gene encoding fGH targetedto the vacuole or the gene encoding GFP targeted to the vacuole werefurther designed to include one of the CPPs as described in the“material and methods” section hereinabove.

Algae lines expressing the vacuole-fGH-MTS-HA or vacuole-fGH-TAT-HA areused to feed fish in feeding trials as described above. Alga linestransformed with the above-constructs wherein the fGH is replaced by GFP(vacuole-GFP-MTS- or vacuole-GFP-TAT-) are used as a model system.Additionally these algae lines are used to feed mice by gavage. At theend of the feeding trials the presence of the algae-expressed protein isexamined in blood of the fish or mice fed with the algae using ELISA orWestern blot analysis directed toward the HA tag. Additionally, GFPfluorescence of blood samples is detected directly by a fluorescenceplate reader or GFP protein is detected by ELISA using an anti GFPantibody.

The results presented above demonstrate that the algae Phaeodactylumtricornutum serves as an efficient means to orally deliver recombinantproteins to various target animals. Without wishing to be bound by anyspecific theory or mechanism of action, the algae serve as a nativebio-encapsulation, with the algae cell wall protecting the recombinantprotein from its enzymatic degradation in the acidic stomach. Theresults further suggest, again without wishing to be bound by anyspecific theory or mechanism of action, that the vacuole targetedprotein enables its efficient absorption from the intestine to thetarget organelle.

In summary, the present application demonstrates for the first time theestablishment of an algae based platform, which enables oraladministration of recombinant proteins to animals in their intact andfunctional form.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

The invention claimed is:
 1. A transgenic eukaryotic microalgacomprising: an expression cassette comprising at least one transcribablepolynucleotide encoding a biologically active exogenous protein whereinthe polynucleotide is operably linked to an expression control sequence,wherein the expression cassette further comprises a vacuole targetingpeptide encoding sequence comprising the nucleic acid sequence of SEQ IDNO:18, encoding the amino acid sequence of SEQ ID NO:4, wherein theexpressed biologically active exogenous protein is localized within themicroalga cell vacuole, and said exogenous protein effects at least oneof growth, development and survival of a target animal.
 2. Thetransgenic microalga of claim 1, wherein the expression cassette furthercomprises a polynucleotide encoding a protein domain that mediate theuptake of the expressed exogenous protein by a xenogeneic cell or axenogeneic tissue.
 3. The transgenic microalga of claim 1, wherein saidmicroalga is a marine alga.
 4. The transgenic microalga of claim 3,wherein said microalga is selected from the group consisting ofPhaeodactylum tricornutum, Dunaliella spp., Nannochloropsis spp.,Nannochloris spp., Tetraselmis spp., Isochrysis galbana, Pavlova spp.,Amphiprora hyaline, Chaetoceros muelleri, and Neochloris oleoabundans.5. The transgenic microalga of claim 1, wherein the biologically activeexogenous protein has a molecular weight of up to 150 kDa.
 6. Thetransgenic microalga of claim 5, wherein the biologically activeexogenous protein has a therapeutic activity.
 7. The transgenicmicroalga of claim 5, wherein the biologically active exogenous proteinis a hormone.
 8. The transgenic microalga of claim 7, wherein thehormone is selected from the group consisting of a growth hormone,appetite inducing hormone and spawning hormone.
 9. An edible compositioncomprising the transgenic microalgae according to claim 1.